Proteins and peptides are vital for biological processes, and recently have attracted significant attention as therapeutic agents. Therapeutic proteins and peptides are known and used in the treatment of many serious diseases, such as diabetes, cancer, HIV, and cardiovascular diseases (3-6). In general, therapeutic proteins and peptides are considered natural, more effective, and less toxic than other drugs, such as synthetic, small molecule drugs.
However, the delivery of therapeutic proteins and peptides is challenging because bioavailability and stability must be maintained during the formulation and sustained release processes. Although there has been intense research in this area, efficacious systems for targeting delivery and sustained release of proteins and peptides in biologically active conformations and concentrations are in need.
A systemic delivery of protein therapeutic agents is feasible, e.g., exenatide for the treatment of type II diabetes (7). But high systemic levels of growth factors can cause unwanted, adverse side effects and immunogenicity, for example, carcinomas and tumor growth (8). The future of therapeutic proteins therefore lies in a focused, local delivery, as in the use of angiogenesis for the treatment of cardiovascular diseases (9).
Therapeutic proteins and peptides pose a problem that small molecule drugs do not encounter, i.e., the need to maintain bioavailability and stability during the formulation process and sustained release. Damage to proteins and peptides can occur, for example, by irreversible conformation changes, denaturation, aggregation, acid- or enzyme-catalyzed degradation, deamidation, and hydrolysis (10-15). In addition, the energy barrier for protein conformation changes in solution also is low, i.e., only 5-20 kcal/mol (16), which is comparable to water-oil interfacial tensions and hydrophobic interactions.
To prevent proteins and peptides from denaturing, an effective sustained release delivery system must meet three main requirements: (a) the formulation process should avoid exposing proteins to organic solvents, water-oil interfaces, crosslinking reagents, and large temperature fluctuations; (b) after administration to an individual, the delivery system should protect proteins and peptides from aggregation, degradation, and conformation changes prior to reaching the targeted tissues; and (c) the drug release profile and pharmacokinetic properties should be well defined and reproducible.
Despite extensive research efforts and diverse technologies developed for a targeted, sustained release of proteins and peptides, few feasible methods of delivery have emerged. Current formulation strategies include four categories: (a) forming solid microspheres or microfibers that encapsulate proteins by emulsion, double emulsion, spray dry, freeze-dry, spray freeze dry, and supercritical anti-solvent processes (17-21); (b) chemical modification, such as PEGylation (22) and acylation (23); (c) forming sustained release depots by a gelling process (24, 25); and (d) delivery by self-assembling peptides and lipids (1, 26, 27). To a certain degree, these protein delivery methods protect proteins from enzymatic degradation and provide a sustained release compared to direct injection.
Encapsulating or entrapping proteins and peptides in biocompatible and biodegradable polymeric systems is one of the most promising routes for a controlled delivery and release of these drugs. Various geometries and configurations of polymeric systems have been reported, including polymeric membranes, matrices, microspheres, and microfibers.
However, each method has its limitations. For example, polymeric membranes have a potential problem of “dose-dumping,” or mass release, of entrapped material due to membrane failure. For potent therapeutic agents, dose-dumping can cause serious problems. Release of proteins also is highly dependent on the microstructure of polymer matrix, such as pore size and density. Therefore, it is difficult to reproduce the release profile for the same protein, and, for different proteins, the release system has to be redesigned.
Solid microsphere and microfiber encapsulated proteins can be delivered by injection or inhalation. The general limitations with this delivery system are (a) low loading efficiency and (b) reduced protein biological activity caused by manufacturing processes involving a high shear rate, exposing proteins to a water-oil interface, and/or high temperature fluctuations (17-21).
Hydrogels potentially are suitable carriers for the delivery of proteins and peptides. Crosslinked networks of water-soluble polymers allow slow diffusion of proteins and peptides. Despite the hydrophilicity of the polymers, the process to encapsulate proteins and peptides into hydrogel can involve UV exposure, high shear rate, exposing proteins to water-oil interfaces, or high temperature fluctuations. More importantly, the release profiles of various proteins depend on the sizes of the molecules and pore size of the hydrogels.
In general, chemical modification, such as PEGylation and acylation, reduces protein degradation and receptor-mediated uptake of the proteins from the systemic circulation (22, 23). However, large proteins have several sites that are accessible to PEGylation or acylation, which produces a high heterogeneity. For example, mono-PEGylated epidermal growth factor (EGF) with PEG 3400 at Lys28 and Lys48 was found to be significantly less active than EGF isomer PEGylated at the amino terminus in an in vitro assay for mitogenic activity (28). In addition, protein conformation and bioavailability need to be further demonstrated for the modified proteins. The disadvantage of gelling depots is that the protein or peptide may be squeezed out of the depot, which results in an initial burst release of the drug (24, 25). Delivery of proteins using lipid and self-assembling peptides has the limitations of low encapsulation rate and low system stability (1, 26, 27).